Analysis of the role of ribonucleases in the regulation of epithelial sheet sealing

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Ribonucleases have been well studied in yeast and bacteria, but their biological significance to multicellular organisms is not well understood. However, there is increasing evidence that specific, timed transcript degradation is critical for many cellular processes, including early development, RNA interference and nonsense-mediated decay. We have used a reverse genetics approach in Drosophila to show that mutations in the 5 prime-3 prime exoribonuclease pacman result in severe defects in thorax closure such that mutants have clefts along the back of the thorax. The dorsal part of the thorax in wild-type Drosophila is formed by cells at the leading edges of the two wing imaginal discs which stretch, migrate towards each other and then fuse along the dorsal midline. This epithelial cell movement is very similar to seen in dorsal closure in Drosophila, hind-brain closure in vertebrates, ventral enclosure in C. elegans and wound healing in humans. Our preliminary experiments have shown that pacman genetically interacts with puckered, a conserved member of the JNK signalling pathway, which is known to be involved in thoracic closure. We have also shown that pacman protein is localised in cytoplasmic foci, which are likely to be specific sites of 5 prime-3 prime mRNA degradation (P-bodies). We propose that pacman plays a key role in epithelial sheet sealing, by targeting particular RNAs. To test this hypothesis we will first use genetic, molecular biology and micro-array analysis to establish whether puckered, or another member of the JNK pathway, is a specific target of the pacman ribonuclease. We will then construct transgenic flies to determine which regions of the target RNA are recognised by the 5 prime-3 prime degradation machinery. Second, we will use confocal microscopy to investigate the intra-cellular localisation of PACMAN to determine whether it is associated with the actin cytoskeleton or other cellular components. Third, we will examine the localisation of PACMAN in relation to other proteins in the 5 prime-3 prime degradation pathway, to determine, as our pilot data suggests, whether PACMAN has a key role in the formation of P-bodies. Finally, we will determine the involvement of PACMAN in wound healing in Drosophila by comparing the wound healing process in mutant, compared to wild-type flies. These innovative studies will shed light on the role of pacman ribonuclease in development.